alexa Development and validation of a serum total testosterone liquid chromatography-tandem mass spectrometry (LC-MS MS) assay calibrated to NIST SRM 971.
Chemical Engineering

Chemical Engineering

Journal of Analytical & Bioanalytical Techniques

Author(s): French D

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Abstract BACKGROUND: At our institution, serum testosterone in adult males is measured by immunoassay while female and pediatric specimens are sent to a reference laboratory for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis due to low concentrations. As this is of significant cost, a testosterone LC-MS/MS assay was developed in-house. METHODS: A 5500 QTRAP® using electrospray ionization and a Shimadzu Prominence with a C18 column were used. Gradient elution with formic acid, water and methanol:acetonitrile at 0.5 ml/min had a 7-min run-time. A liquid-liquid extraction with hexane:ethyl acetate was carried out on 200 μl of serum. Multiple reaction monitoring was employed. RESULTS: Sample preparation took ~80 min for 21 samples. Six calibrators were used (0-1263 ng/dl; concentration assigned by NIST SRM 971) with 3 quality controls (9, 168 and 532 ng/dl). The limits of detection and quantitation were 1 and 2 ng/dl respectively. Extraction recovery was ~90\% and ion suppression ~5\%. Within-run and total precision studies yielded <15\% CV at the limit of quantitation and <7\% CV through the rest of the linear range. Isobaric interferences were baseline separated from testosterone. Method comparisons between this assay, an immunoassay, and another LC-MS/MS assay were completed. CONCLUSIONS: An accurate and sensitive LC-MS/MS assay for total testosterone was developed. Bringing this assay in-house reduces turnaround time for clinicians and patients and saves our institution funds. Copyright © 2012 Elsevier B.V. All rights reserved. This article was published in Clin Chim Acta and referenced in Journal of Analytical & Bioanalytical Techniques

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